This proposal brings together a unique multidisciplinary team of leading experts in alcohol studies and real time dynamic fluorescence imaging of cells and subcellular structures in intact tissues, high resolution electron tomography, sophisticated molecular biology and computational biology to address a fundamental question of how a deregulation of local interactions between organelles can result in global cell dysfunction, tissue damage and disease. The study addresses the regulation of the mitochondria-endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR) interface and its relationship to localized Ca2+ signaling, formation of reactive oxygen species (ROS) and ER stress. The central hypothesis to be tested is that chronic alcohol exposure causes impaired ER/SR-mitochondrial structural and functional coupling and thereby increases the susceptibility to cell injury in liver and skeletal muscle. Our group has developed methods to manipulate the ER/SR-mitochondrial interface and has shown that this has consequences for localized Ca2+ signaling. Our first aim is to further develop these new experimental tools to be able a) to manipulate and measure the ER/SR-mitochondrial interface, b) to monitor the impact of controlled mitochondrial localization on local [Ca2+] signals and local ROS formation and c) to assess ER/SR stress responses in real time at the single cell level. This will help us analyze the relationship between ER/SR stress and changes in localized Ca2+ and ROS signaling. We will then test the concept that the effect of chronic alcohol exposure on ER/SR-mitochondrial interactions leads to impairments in ER/SR-mitochondrial structure, calcium signaling and induces ER/SR- mitochondrial stress responses in the liver and skeletal muscle. Finally, we will determine whether the alterations in ER-mitochondrial morphology and function contribute to alcohol-induced metabolic dysfunction and cell injury in vivo. With these approaches, we can answer fundamental questions about the subcellular organization of the ER-mitochondrial network and its disruption by ethanol. These studies will shed new light on the mechanisms by which alcohol abuse can cause tissue damage and will provide unique opportunities for the development of innovative treatment strategies. PUBLIC HEALTH RELEVANCE: Chronic alcoholism is associated with ER stress and mitochondrial dysfunction and changes in ER and mitochondrial morphology in multiple tissues. The ER/SR-mitochondrial junctions are important for the function and structure of both ER/SR and mitochondria and are envisioned as a target and an important mediator of the alcohol's effect on liver and skeletal muscle injury. A multidisciplinary approach utilizing expertise in alcohol studies and real time dynamic fluorescence imaging of cells and subcellular structures in intact tissues, high resolution electron tomography, sophisticated molecular biology and computational biology will help to address a fundamental question of how a deregulation of local interactions between organelles can result in global cell dysfunction, tissue damage and disease.